羊草脂氧合酶同源蛋白基因 LcLHP 的克隆与表达分析

摘要

In order to know the relationships between Lipoxygenase and salt stress,a full length cDNA named LcLHP related to saline-alkali tolerance was cloned from Leymus chinensis by RT-PCR and RACE(Rapid amplifica-tion of cDNA ends).LcLHP gene (GenBank:KJ472894)with 537 bp open reading frame encoded a protein with a predicted length of 1 78 amino acids.LcLHP has PLAT /LH2 structural domain,which is induced by stress.Homolo-gy analysis showed that the deduced amino acid sequence of LcLHP shared high homology with PLAT /LH2 protein family of Spartina alterniflora and PLAT stress protein.Protein function prediction showed N terminal of LcLHP may have signal peptide,act as enzyme.The expression of LcLHP was analyzed by RT-PCR under salinity-alkalinity stressed condition.With the extension of the salinity-alkalinity stressed time,the expression of LcLHP increased from 0 to 1 2 h in the leaf,and then decreased,the peak of expression level arrived at 1 2 h.Construction of pGEX-LcLHP ex-pression vector,IPTG induced prokaryotic expression,we got GST-LcLHP fusion protein and verified by Western Blot.%为验证羊草脂氧合酶同源蛋白基因 LcLHP 与盐胁迫的关系，利用 RACE（Rapid amplification of cDNA ends）克隆技术从羊草中克隆得到抗盐碱相关基因脂氧合酶同源蛋白基因（LHP，Lipoxygenase homology protein）的 cDNA 全长序列，其 GenBank 登录号为 KJ472894。LcLHP 基因开放阅读框为537 bp，编码178个氨基酸。LcLHP 中含有 PLAT ／LH2结构域，该结构域蛋白家族的许多成员受胁迫诱导。同源性分析显示，LcLHP 与互花米草 PLAT ／LH2家族蛋白脂氧合酶、PLAT 植物胁迫结构域包含蛋白等同源性较高，蛋白功能预测显示其 N 端可能存在信号肽并可能具有酶功能。RT-PCR 分析显示，在一定盐碱胁迫条件下，随着处理试剂 Na2 CO3溶液处理时间的延长，LHP 基因在羊草中的表达量呈单峰趋势，在12 h 时达到峰值；构建重组表达载体 pGEX-LcLHP 并进行 IPTG 原核诱导表达培养，获得了 GST-LcLHP 融合蛋白并用 GST 抗体进行 Western Blot 试验得到验证。